Inhibition of Lin-11, Isl-1, and Mec‑3 Kinase (LIMK) Activity Attenuates Astrogliosis and Vascular Remodeling in an Experimental Model of Cuprizone-Induced Demyelination in Mice

Abstract

A variety of neurological impairments are caused by the loss of myelin, which is a hallmark of demyelinating diseases such as multiple sclerosis (MS). The cuprizone model is a well-established demyelination model in which specific brain regions undergo persistent demyelination after cuprizone administration. LIM kinases (LIMKs), a downstream of the RhoA/ROCK pathway, regulate cytoskeletal reorganization through the depolymerization of actin filaments. In our previous findings, LIMK1’s phosphorylation was found to be enhanced during chronic phases of spinal cord injury (SCI), leading to scar formation and neuronal loss. However, the specific implications of the LIMK pathway on demyelination and axonal damage in nontraumatic neurodegenerative disorders remain unresolved. In this study, we explored the differential expressions of phosphorylated LIMK1 (p-LIMK1) in a cuprizone-induced acute and chronic demyelination model and its further correlation with astrogliosis and vascular remodeling. BMS-5, the LIMK inhibitor, alleviated cuprizone-induced demyelination, exerting its therapeutic effects via inhibiting LIMK expression. This resulted in the diminution of astrogliosis marked by reduced glial fibrillary acidic protein (GFAP) and neural/glial antigen 2 (NG2) expression and the subsequent maintenance of myelin integrity. Additionally, BMS-5 treatment was associated with improved motor function. Overall, these findings suggest that LIMK inhibition with BMS-5 holds promise as a therapeutic approach for demyelination.

Demyelinating diseases, including MS, are characterized by the loss of myelin, which in turn produces a range of neurological impairments in adulthood. Inflammation, oligodendrocyte death, and axonal degeneration are pathological processes that contribute to neurological impairment in demyelinating diseases. It is a multifaceted and diverse disorder that may present itself in numerous forms characterized by the immune system erroneously targeting the myelin sheath. At the histopathological level, they are distinguished by localized loss of myelin in both white and gray matter, which are brought about by interactions between brain cells (such as microglia and astrocytes) and immune cells from outside the brain (such as lymphocytes and monocytes). Damage to nerve cells occurs as a consequence of demyelination as well as localized and diffuse invasion and (re)­activation of these immune cells.

The cuprizone (bis-cyclohexanone oxaldihydrazone) model is a well-established model of toxic demyelination, where the administration of cuprizone leads to the consistent demyelination. Within a few days of oral intoxication with the copper-chelator cuprizone, oligodendrocytes undergo apoptosis without a marked lymphocytic response. This is promptly followed by the activation of the brain’s innate immune cells, specifically microglia and astrocytes, and ultimately results in the demyelination specifically in corpus callosum (CC), superior cerebellar peduncles, and hippocampus. These pathological characteristics resemble progressive MS lesions. Reactive astrocytes play a crucial part in demyelinating diseases as early microglial activation sets off a series of signals that induce astrocytes to produce signaling molecules that lead to demyelination. Studying the remyelination phase becomes much easier when the demyelinating substance, cuprizone, is removed. Cuprizone causes effective spontaneous remyelination after a single incident of acute demyelination when administered as a short-term treatment. Extended use of cuprizone leads to a persistent state of demyelination, which then undergoes inadequate remyelination. ,

Myelin-associated axonal growth inhibitors, such as myelin-associated glycoprotein (MAG), Nogo, myelin oligodendrocyte glycoprotein (MOG), and repulsive guidance molecule, restrict the development of axons by activating the Ras homologue family member A (RhoA)/Rho-associated coiled-coil containing protein kinase (ROCK) pathway. ,, Downstream of this RhoA/ROCK pathway, LIMKs are involved in the regulation of cytoskeletal reorganization. With two isoforms, LIMK1 and LIMK2, LIMK proteins exhibit varied expression in diverse tissues with their expression being controlled via separate mechanisms. They are activated by certain signaling pathways, and their activity is then regulated in accordance with these pathways. Though LIMK2 has widespread expression, LIMK1 is mostly localized in the central nervous system (CNS), embryonic and adult tissues, and to a lesser extent in the heart and skeletal muscle. Increased expression of LIMK1 in dorsal root ganglion (DRG) neurons was found to be responsible for the inhibition of growth cone movement and the development of neurites. Additionally, LIMK was found to be involved as a downstream of vascular endothelial growth factor (VEGF) in growth cone stimulation, and the growth cone motility was increased by VEGF induction through the cytoskeletal rearrangement activity driven by LIMK. Hence, VEGF was identified as a positive regulator of growth cone motility through the LIMK pathway. We previously investigated the correlation between major glial and neuronal markers and the differential expressions of p-LIMKs in neurodegeneration associated with SCI. The phosphorylated form of LIMK1 appeared to be increased during the chronic stages of injury when scar formation and neuronal loss were pronounced. Eventually, the LIMK pathway drew our attention, and hence we used its specific inhibitor BMS-5, which demonstrated neuroprotective effects after SCI.

However, the precise impact of the LIMK pathway on the nontraumatic model of demyelination remains uncertain. Hence, herein we investigated the differential expressions of p-LIMK1 in cuprizone-induced acute and chronic demyelination and its further correlation with astrogliosis and vascular remodeling. Once the expression pattern of p-LIMKs had been determined, we investigated their inhibitory effect using BMS-5, in both cuprizone-induced demyelination and remyelination.

Results

Acute Cuprizone Administration Induces Motor Incoordination, Neuronal Cell Loss, and Demyelination

Motor coordination was assessed using a rota-rod apparatus. All animals enhanced their abilities and acquired the skill to maintain balance on the rota rod. Prior to cuprizone feeding, there was no discernible difference in the latency to fall between the two groups. However, with the progressing time period, the animals in the cuprizone-treated group showed a significant decrease in the latency to fall compared to the control group (*p < 0.05 vs control) (Figure A). Following the cessation of cuprizone administration at week 5, the animals showed a trend toward motor recovery, as evidenced by a gradual increase in the latency to fall by week 7. Then, we performed Nissl staining to confirm the histomorphological alterations induced by cuprizone in the hippocampus. The results of the Nissl staining revealed that the control group exhibited strong Nissl intensity in the clearly defined neuronal soma in the hippocampus. However, the cuprizone-treated groups displayed sparsely expressed degenerating neurons with the most significant changes in week 5 (**p < 0.01 vs control) (Figure B, C). Subsequently, our attention was directed to the CC area, which is extensively investigated in this particular model of demyelination. As the largest white matter structure in the brain, the CC links the two hemispheres’ homologous cortical regions and is essential for transmitting information related to motor control, cognition, and perception. To analyze the loss of myelin in the CC of cuprizone-fed mice, Luxol Fast Blue (LFB) staining was performed. In the control group of mice, the CC exhibited a high level of myelination, as shown by the rectangular box (Figure D). Notable demyelination was observed in the mice given the cuprizone diet, especially in week 5 (Figure D), but this effect was somewhat reversed after 2 weeks of cuprizone withdrawal.

1.

The acute exposure of cuprizone causes motor incoordination, loss of neuronal cell bodies, and myelin as shown by behavioral testing and histological assessment. A) Motor incoordination was assessed by using the rota rod. The graph shows the latency to fall in two different groups: control and cuprizone-administered mice. B) Representative images of coronal sections of mice brain stained by cresyl violet revealed that cuprizone-treated groups displayed sparsely expressed degenerating neurons whereas the control group exhibited strong Nissl intensity in the clearly defined neuronal soma in the hippocampus. C) Quantification of Nissl integrated density in different groups. D) Representative images of LFB staining of CC for control, week 1, 3, 5 of demyelination, and week 7 of remyelination after cuprizone exposure. Data are expressed as mean ± SEM. Differences between groups are expressed as *p < 0.05, **p < 0.01 vs control.

Chronic Administration of Cuprizone Leads to Motor Impairment along with a Reduction in Neuronal Cell Bodies and Myelin

The animals, after chronic administration of cuprizone in weeks 10 and 12, exhibited a significant reduction (****p < 0.0001 vs control) in the time spent in rota rod, as compared to the control group. However, in the case of cuprizone withdrawal at the remyelination period (week 14), the latency to fall did not show any notable alteration compared to the previous demyelination group (Figure A). Hence, this finding reveals that the withdrawal of cuprizone was inadequate to restore motor coordination after a chronic exposure (up to week 12). The results of the Nissl stain showed that chronic intoxication of cuprizone had a significant detrimental effect on the neuronal cells in the hippocampus of the experimental animals (***p < 0.001 vs control) (Figure B, C). While observing the changes in myelin in the CC region of the brain, the groups of weeks 10, 12, and 14 with chronic cuprizone exposure were found to have a visible loss in myelin compared to the control group (Figure D).

2.

The chronic exposure of cuprizone causes motor incoordination, loss of neuronal cell bodies, and myelin as shown by behavioral testing and histological assessment. A) Motor incoordination was assessed by using the rota rod. The graph shows the latency to fall in different groups. B) Representative images of coronal sections of mice brain stained by cresyl violet revealed that cuprizone-treated groups displayed sparsely expressed degenerating neurons whereas the control group exhibited strong Nissl intensity in the clearly defined neuronal soma in the hippocampus. Directional arrows point to specific zones of neuronal loss and layer attenuation in the CA1 and DG. C) Quantification of Nissl integrated density in different groups. D) Representative images of LFB staining of corpus callosum for control, week 7, 10, 12 of demyelination, and week 14 of remyelination after cuprizone exposure. Data are expressed as means ± SEM. Differences between groups are expressed as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control.

Phosphorylated LIMK1 Upregulation and Subsequent Expressions of Endothelial Markers Correlate with Acute Cuprizone-Induced Demyelination and Remyelination

The expression of p-LIMK1 was assessed along with vascular and oligodendrocytic markers in the brain hippocampus through Western blotting at different time points after acute cuprizone exposure. We found a significant upregulation of p-LIMK1 at week 5 of demyelination and week 7 of remyelination after cuprizone administration (**p < 0.01 vs control) (Figure A, B). Administering cuprizone induces apoptosis of oligodendrocytes, resulting in eventual demyelination. We found a loss of oligodendrocytes, though not statistically significant, along with acute time points of demyelination (Figure A, B). Next, we aimed to assess vascular remodeling through the expression of VEGF after acute cuprizone exposure. Research suggests that VEGF plays a role in the pathology of MS. VEGF-A is mostly produced by astrocytes and neurons, but it is also released by cerebral endothelial cells and leukocytes. Here, we found a significant downregulation of VEGF at week 5 of demyelination (*p < 0.05 vs control) and then an increase in the expression at week 7 of remyelination (Figure A, B). It is reported that during the late phase of MS, the levels of VEGF, which serves as a protective agent for neurons and neural progenitors, are reduced in the cerebrospinal fluid of MS patients. This reduction is also seen in peripheral blood mononuclear cells from individuals with secondary progressive MS. In our observations, the downregulation of VEGF at demyelination justifies the previous findings. Next, we assessed the expression of vascular cell adhesion molecule-1 (VCAM-1) after acute cuprizone exposure. Here, significant upregulations were found at weeks 1 and 3 of demyelination (***p < 0.001 vs control) (Figure A, B). Further, we checked the expression of established pathophysiological markers by immunohistochemistry (IHC) to correlate them with the expression of p-LIMK1. As p-LIMK1 was found to be upregulated with increasing time points after acute cuprizone exposure, concurrently, myelin basic protein (MBP) showed a reduced expression with the most significant reduction at week 5 of demyelination (***p < 0.001 vs control) (Figure C, D). In Figure E, we showed prolonged astrocytic activity with a significant upregulation of the reactive astrocytic marker GFAP at week 5 of acute demyelination (**p < 0.01 vs control). Astrogliosis may result in the accumulation of extracellular matrix molecules, leading to the formation of a glial scar. This scar has been shown to be associated with chronic types of MS and is believed to hinder the recruitment and/or differentiation of oligodendrocyte progenitors after demyelination. A significant increase in the expression of NG2 at week 3 and week 5 of demyelination was observed in our findings (****p < 0.0001 vs control) (Figure E, F), which confirms the impediment of axonal growth.

3.

The increased expression of p-LIMK following acute cuprizone exposure results in vascular remodeling, astrogliosis, and myelin loss. A) Representative Western blot images of p-LIMK1, OLIG 2, VEGF, and VCAM for control and at different time points of acute cuprizone exposure. B) Quantification of p-LIMK1, OLIG 2, VEGF, and VCAM, respectively. C) Representative IHC images of MBP in the brain hippocampal regions at weeks 1, 3, 5 of demyelination and week 7 of remyelination. D) Quantification of MBP indicates a significant reduction of MBP in demyelination (week 1, 3, 5) and its recovery during remyelination (week 7). E) Representative IHC images of GFAP and NG2 in the brain hippocampal region (Scale bar: 200 μm). F) Quantification of fluorescence intensity for NG2 and GFAP demonstrated prolonged astrocytic activity with a significant upregulation of NG2 at weeks 3 and 5 of demyelination. Data are expressed as means ± SEM. Differences between groups are expressed as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control.

Phosphorylated LIMK1 Expression Is Not Associated with Astrogliosis and Myelin Loss at Chronic Time Points of Cuprizone Administration

We again investigated the expressions of p-LIMK1 after chronic cuprizone exposure and aimed to correlate with vascular remodeling, astrogliosis, and myelin loss. Here, we did not find any significant change in the expressions of p-LIMK1 at chronic time points (Figure A and B). Though at week 10 it showed a downregulation, however, it was not statistically significant. At the remyelination of week14, it almost restored to the normal level. In case of OLIG 2 and VEGF also, we could not find any significant alteration throughout the time points. However, VCAM was found to be significantly upregulated at week 7 (**p < 0.01 vs control) and week 10 of demyelination and restored almost to the normal level at week 14 of remyelination. Further, we analyzed the expressions of MBP, GFAP, and NG2 by IHC. MBP was found to be significantly downregulated as compared to the control at all chronic demyelination time points (weeks 7, 10, and 12) (***p < 0.001 vs control). While a partial recovery of MBP levels was observed at week 14 following cuprizone withdrawal, expression remained significantly below control levels. This suggests that spontaneous remyelination initiates at week 14 after cuprizone withdrawal, but withdrawal alone is not sufficient to achieve full restorative myelination within this time frame (Figure C, D). The observation was consistent with the findings of astrogliosis as well. Astrocytic reactivity was observed following chronic cuprizone administration with significantly increased GFAP expression at week 12 of demyelination (*p < 0.05 vs control). A significant upregulation of NG2 (**p < 0.01 vs control) was found at all time points of demyelination and remyelination, suggesting that oligodendrocyte precursor cells (OPCs) were recruited to the demyelinated lesions but fail to mature into myelinating oligodendrocytes (Figure E, F).

4.

The expressions of p-LIMK, OLIG 2, VEGF, and VCAM at different time points after chronic cuprizone exposure. A) Representative Western blot images of p-LIMK1, OLIG 2, and endothelial markers (VEGF, VCAM) for control and different time points of chronic cuprizone exposure. B) Quantification of phosphorylated LIMK1, OLIG 2, VEGF, and VCAM, respectively. C) Representative IHC images of MBP in brain hippocampal regions at weeks 7, 10, 12 of demyelination and week 14 of remyelination. D) Quantification of MBP fluorescence intensity indicates significantly reduced MBP expression in chronic demyelination (week 7, 10, 12) and its recovery during remyelination (week 14). E) Representative IHC images of GFAP and NG2 in the brain hippocampal region (Scale bar: 200 μm). F) Quantification of fluorescence intensity for NG2 and GFAP demonstrated prolonged astrocytic activity with a significant upregulation of NG2 at weeks 7, 10, and 12 of demyelination and week 14 of remyelination. *p < 0.05, **p < 0.01, ***p < 0.001 vs control.

LIMK Inhibition Corresponds to an Improved Motor Coordination and Maintains Hippocampal Histomorphology after Acute Cuprizone Exposure

As we found that there was a significant upregulation of p-LIMK1 at week 5 of demyelination and week 7 of remyelination after acute exposure of cuprizone, we decided to target LIMK only at acute study and check its effect to promote myelination with its specific inhibitor, BMS-5. In the rota-rod test, BMS-5 showed a significant enhancement in the time spent as compared to the cuprizone-administered group at week 5 of demyelination. Though at week 3 of demyelination and week 7 of remyelination, the BMS-5-treated group showed an increase in the time spent, but it was not statistically significant (Figure A). Findings from the Nissl staining showed high Nissl intensity in the hippocampus of BMS-5-treated groups after acute cuprizone administration (**p < 0.01 vs control, #p < 0.05 vs respective demyelination or remyelination group) (Figure B and C).

5.

LIMK inhibition leads to improved motor coordination and maintains the morphology of hippocampal neurons after acute cuprizone exposure. A) Representative graph showing motor coordination assessment in control, cuprizone, and BMS-5-treated groups at weeks 1, 3, and 5 of demyelination, and week 7 of remyelination after cuprizone administration. B) Representative images of Nissl staining in weeks 3, 5, and 7 and their respective BMS-5-treated groups. C) Quantification of Nissl integrated density in the aforementioned groups. Data are expressed as means ± SEM. Differences between groups are expressed as *p < 0.05 vs control, **p < 0.01 vs control, #p < 0.05 vs their respective demyelination and remyelination groups.

LIMK Inhibition Promotes Myelination and Attenuates Astrogliosis after Acute Exposure of Cuprizone

We found a significant downregulation in the expression of p-LIMK1 in the BMS-5-treated group at week 7 of remyelination (#p < 0.05 vs week 7 remyelination group) (Figure A, C). Though at week 3 and week 5 of demyelination also, BMS-5-treated group showed a reduction in the expression of p-LIMK1, it was not statistically significant. Furthermore, we checked the effect of LIMK inhibition in demyelination. BMS-5-treated groups were found to promote myelination through an enhanced expression of MBP in the demyelination phase of acute cuprizone exposure (#p < 0.05 vs week 5 demyelination group) (Figure B, E). Then, we investigated the effect of BMS-5 in astrogliosis. Though BMS-5 did not show any notable change in the expression of GFAP, but it showed a promising effect through diminished expression of NG2 in both demyelination and remyelination time points of acute cuprizone administration (###p < 0.001 vs week 5 of demyelination, #p < 0.05 vs week 7 of remyelination) (Figure D, F). To validate our IHC findings, Western blot analysis was performed for GFAP across weeks 3, 5, and 7 in cuprizone-treated and control groups, alongside the treatment group receiving BMS-5. It showed a nonsignificant increasing trend at week 5 (Supplementary Figure S4), consistent with the localized reactivity observed in Figure .

6.

Following an acute exposure to cuprizone, LIMK inhibition attenuates astrogliosis and fosters myelination. A) Representative Western blot images of different groups (control, weeks 3, 5 of demyelination and 7 of remyelination) along with BMS-5 treatment. B) Quantification of fluorescent intensity of MBP demonstrated that BMS-5 treatment may promote myelination by enhancing MBP expression during the demyelination phase of acute cuprizone exposure (weeks 3 and 5). C) Quantification of p-LIMK with respect to the loading control GAPDH in different groups. D) Quantification of fluorescence intensity for NG2 and GFAP at different time points. BMS-5 reduced NG2 expression in both demyelination and remyelination time points of acute cuprizone administration, despite no significant change in GFAP expression. E) Representative IHC images showing MBP expression. F) Representative IHC images of GFAP and NG2 during demyelination and remyelination along with BMS-5-treated groups (Scale bar: 200 μm). Data are expressed as means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 vs control, #p < 0.05, ##p < 0.01, ###p < 0.001 vs their respective demyelination and remyelination groups).

Discussion

The cuprizone model is a comprehensive and easily applicable method for studying several aspects of MS pathophysiology. The cuprizone-induced pathology effectively reproduces several aspects of progressive MS clinical characteristics. In 1998, Hiremath and colleagues developed a protocol for administering cuprizone at a lower dosage (0.2% w/w) to C57BL/6 mice. This approach has since been extensively adopted and is still in use today. Cuprizone is often administered to mice for a period of 4–6 weeks in order to investigate acute demyelination or for a period of up to 12 weeks to investigate chronic demyelination. In addition, mice are occasionally monitored following cuprizone removal to study remyelination. When administered to rodents, the copper chelator cuprizone causes a depletion in myelin and oligodendrocytes in certain areas of the brain while causing minimal disruption to the blood–brain barrier (BBB) and peripheral immune cell infiltration. As oligodendrocytes are highly specialized cells that produce myelin sheaths, they require a high energy supply from mitochondria to maintain the integrity of myelin sheaths. , Being a copper chelator, cuprizone in turn prevents the activity of copper-dependent mitochondrial enzymes, namely cytochrome oxidase and monoamine oxidase. Hence, a disruption in the energy metabolism of oligodendrocyte mitochondria by cuprizone leads to apoptosis in the oligodendrocytes and, eventually, demyelination. Research has shown that oligodendrocyte apoptosis is initiated within the first week of cuprizone treatment. Activated caspase 3 became apparent in apoptotic oligodendrocytes during the initial phase of the cuprizone diet (days 6 and 10), but not in later stages (day 21). − The early onset of motor impairment observed at week 1 in our study may be attributed to acute metabolic stress and mitochondrial dysfunction induced by cuprizone.

RhoA is a versatile small GTPase protein that plays a crucial role in regulating OPC development and oligodendrocyte maturation. RhoA/ROCK controls the rearrangement of the cytoskeleton by stabilizing actin fibers, which in turn affects the morphological changes required for the differentiation of OPCs and the maturation of oligodendrocytes. Activation of the RhoA/ROCK signaling axis was found to hinder OPC differentiation, while inactivation of RhoA/ROCK promoted OPC differentiation in vitro. This impact of RhoA/ROCK inactivation was also observed in multiple conditions, including in the presence of myelin, after SCI, and hypoxia in mice. Another study reported that pharmacological inactivation of ROCK was capable of resolving abnormalities in oligodendrogenesis and functional CNS myelination that occurred in cannabinoid CB1 receptor-lacking OPCs throughout postnatal development in mice. Furthermore, Y-39983, a specific ROCK inhibitor, was found to be protective against demyelination and axonal damage in an animal model of chronic experimental autoimmune encephalomyelitis (EAE). In addition to raising the level of myelin proteins, Y-39983 considerably reduced EAE clinical symptoms and prevented recurrence. However, three primary substrates of ROCK in axons, namely phosphorylated myosin light chain kinase (p-MLC), p-LIMK, and phosphorylated collapsing response mediator protein 2 (p-CRMP2), all participate in the reorganization of the cytoskeleton, specifically actin depolymerization. Hence, the suppressive impact of Y-39983 on demyelination might be attributed to its ability to hinder cytoskeletal reorganization in axons by inhibiting p-MLC, p-LIMK, and p-CRMP2 altogether. Since ROCK is a major regulator of several other kinases, it would be inappropriate to assume that inhibiting ROCK is solely responsible for the impact of ROCK inhibition. Hence, in this study, we evaluated the pathophysiology of acute and chronic cuprizone-induced demyelination by focusing on LIMK, a specific downstream target of ROCK.

After the preliminary validation of the cuprizone model through behavioral and histomorphological analysis, we checked the expression patterns of p-LIMK1 in both acute and chronic cases of demyelination and remyelination. A significant upregulation of p-LIMK1 was found at week 5 of demyelination and week 7 of remyelination after cuprizone administration; however, no such significant alteration was found at chronic time points of demyelination and remyelination after cuprizone exposure. The significant upregulation of p-LIMK1 specifically at week 5 suggests that the RhoA/ROCK/LIMK pathway serves as a primary driver of active cytoskeletal remodeling and glial reactivity during the acute phase of demyelination. A study by Goldshmit et al. established that RhoA activation was critical for the initial transition of astrocytes to a reactive phenotype, leading to the glial scar. The return of p-LIMK1 to basal levels during the chronic phase, despite persistent GFAP and NG2 expression, suggests a temporal dissociation between the process of glial reactivity and its end-products. Different ROCK-downstream effectors, like myosin phosphatase target subunit 1 (MYPT1), can maintain different aspects of the chronic scar even when LIMK activity drops. This is supported by the fact that chronic stress stimulated the phosphorylation of MYPT1, an exclusive target of RhoA-ROCK2. This phosphorylation inhibited MLC phosphatase activity, promoting actomyosin contractility and a subsequent reduction in dendritic spine size and density. Other studies revealed that astrogliosis persisted under chronic cuprizone treatment, but the number of astrocytes remained same with that of the acute stage. , This implies a temporal shift in the glial response, whereas the acute phase is characterized by a peak in molecular signaling and the chronic stage is marked by a failure of many established acute signaling pathways. In a chronic neuropathic pain animal model induced by sparing nerve injury (SNI), the phosphorylated form of LIMKs (p-LIMK) showed a considerable rise 1 day after SNI. It reached its highest level on day 3 and reverted to its basal level 10 days after SNI. Unlike the SNI model, where p-LIMK levels peaked 1–2 days after surgery, plantar incisions led to a rapid rise in LIMK phosphorylation within 0.5 to 6 h after the incision procedure in the postsurgical pain (PSP) model. Notably, the intrathecal administration of a single dose (5 μg) of a particular LIMK inhibitor, LIMKi-3/BMS-5, during the activation period had a crucial impact. It successfully hindered the process of central sensitization and greatly reduced or postponed the occurrence of pain-related behaviors in both SNI and PSP mice.

The vascular remodeling as evident by decreased VEGF expression and the existence of VCAM-1 could potentially suggest an impairment in the neurovascular unit along with peripheral leukocyte infiltration into the CNS. A study by Berghoff et al. revealed that the onset of persistent BBB impairment precedes demyelination. Astrocytes, rather than microglia, were found to play a pivotal role in recruiting the pro-inflammatory cytokines and chemokines during the early stages of the cuprizone model, ultimately leading to the downregulation of BBB maintenance factors. Likewise, the early histological abnormalities that we found in the CC are more likely due to the initial vascular endothelial stress, as demonstrated by our VCAM-1 data. In the chronic time points of our model (weeks 7–14), the absence of significant alterations in OLIG 2 and VEGF suggests a state of regenerative plateau, where the endogenous repair mechanisms are no longer sufficient to counteract the metabolic insult. The study by Lindner et al. showed that under 12 weeks of cuprizone administration, macrophage/microglial accumulation peaked at week 6 and subsequently persisted at a reduced level throughout the treatment period. During the recovery phase, these populations remained detectable at lower levels until the termination of the study. However, the sustained expression of VCAM-1 during these same time points suggests that the BBB remains in a pro-inflammatory state, facilitating lymphocyte recruitment. These observations are supported by the findings in cortical lesions of MS patients, which exhibited an enhanced cytokine and chemokine expression by resident astrocytes and microglia. This inflammatory profile facilitated peripheral leukocyte recruitment through the induction of endothelial adhesion molecules.

With regard to the molecular basis of MBP downregulation, it has already been shown in a lysophosphatidylcholine-induced model of sciatic nerve injury that significant demyelination along with downregulation of MAG hindered nerve sprouting by controlling actin assembly through the Rho-ROCK-LIMK pathway. Consistent with our previous finding, where we showed that p-LIMK induced glial activation through the phosphorylation of its downstream protein cofilin at chronic phases in a rat contusion model of SCI, here also we found a profound astrogliosis at both acute and chronic demyelination, which could potentially suggest its role as a barrier to oligodendrocyte differentiation and subsequent myelin loss.

Determining that an active (phosphorylated) form of LIMK1 exacerbated the demyelination in the acute phase of the cuprizone-induced model, we opted to employ the commercially available inhibitor, BMS-5, targeting it. In our previous study, targeting LIMK1 with BMS-5, and thereby inhibiting cofilin phosphorylation, was likely responsible for the neuronal structure remodeling. A single dose of BMS-5 (1 mg/kg), given just after the SCI, was found to be beneficial via modulation at the cytoskeletal level. In another study, we demonstrated that the same dose of BMS-5 reversed neuronal loss in an MPTP-induced Parkinson’s disease model. This neuroprotective effect was accompanied by the restoration of tyrosinated tubulin levels, further underscoring the critical role of the LIMK in microtubule stabilization. Consistent with our previous findings, the same dose of BMS-5 was administered as a prophylactic single dose at Day 0. This approach allowed us to examine the attenuation of acute signaling at weeks 3 and 5, as well as its downstream influence on the remyelination once the cuprizone was withdrawn. BMS-5 showed a notable downregulation of p-LIMK1 in the acute phases, significantly at week 7 of remyelination. Behavioral analysis and histological findings in our study revealed that BMS-5 had a promising effect improving motor function and restoring the integrity of neuronal cells. Findings regarding the expression of MBP in BMS-5-treated groups justify this observation. We observed a significant increase in NG2 expression following cuprizone administration, indicating a robust recruitment of oligodendrocyte precursors to the demyelinated zones. However, the persistence of these NG2+ cells at week 5 suggests a failure in their transition to mature oligodendrocytes, a process that was partially rescued by BMS-5 treatment.

Conclusion

Overall, our study demonstrates that phosphorylated LIMK1 is significantly upregulated in the acute phase of the cuprizone-induced demyelination model, and its enhancement correlates with astrogliosis, remodeling in vascular markers, and loss of myelin. Simultaneously, inhibition of LIMK with BMS-5 may exhibit a novel therapeutic strategy to alleviate p-LIMK1-induced complexities in nontraumatic demyelination.

Materials and Methods

Model Induction

All animal experiments were performed following the protocols evaluated and approved by the Institutional Animal Ethics Committee (IAEC NO: IAEC/2023/028). The IAEC’s standards and instructions were closely followed following the procurement of the animals. Adult female C57BL/6 mice, weight ranging from 18 to 22 g, were housed in a controlled environment (12 h light/dark cycle) and provided with food and water ad libitum. A total of 136 mice were utilized across three experimental phases (acute, chronic, and therapeutic). The detailed breakdown of animal allocation and experimental distribution has been described in the Supplementary Table S1. For the induction of CNS inflammation and demyelination, cuprizone (C9012, Sigma-Aldrich) was supplemented with standard rodent chow (0.2% w/w). To induce acute demyelination, mice were fed with cuprizone for 5 weeks and then monitored for 2 weeks for remyelination. For chronic demyelination, mice were treated with cuprizone for 12 weeks and then left to remyelinate for a further 2 weeks with normal chow.

Administration of the Compound

The LIM kinase inhibitor BMS-5/LIMKi-3 was purchased from Cayman Chemicals (21072). The stock solution was made using dimethyl sulfoxide (DMSO). A prophylactic single dose of BMS-5 (1 mg/kg, i.p.) was administered on Day 0, followed by a 5 week 0.2% cuprizone diet to induce acute demyelination. Following this 5 week period, cuprizone was withdrawn for 2 weeks (weeks 6–7) to evaluate the impact of the initial LIMK inhibition on the spontaneous remyelination phase.

Behavioral Assessment

Motor coordination was evaluated using the rota-rod test by measuring the time spent on the rod. The rota-rod apparatus was set to gradually accelerate from 4 to 40 rpm, and the latency to fall was recorded as a measure of motor coordination impairment. The parameters such as speed, duration of cuprizone exposure, noise, and light exposure were carefully monitored to ensure reproducibility and reliability of the results. Mice were tested for 3 consecutive trials with a 10 min intertrial interval. Statistical analysis was performed to assess the differences among the experimental groups and the control.

Histological Analysis

At different time points after cuprizone exposure (week 1, 3, 5, 7, 10, 12, 14), mice were deeply anaesthetized via an intraperitoneal injection of a 100 mg/kg ketamine and a 10 mg/kg xylazine cocktail, transcardially perfused with 4% paraformaldehyde. The brains were postfixed for 24 h in 4% paraformaldehyde. A sucrose gradient was used to freeze-protect the fixed brains. Consecutively, 30 μm thick cryosections were taken using a cryostat. The tissue sections were processed for Nissl and LFB staining. For Nissl staining, sections were rehydrated and stained with cresyl violet solution. For LFB staining, sections were incubated in LFB (L0294, Sigma-Aldrich) and the manufacturer’s protocol was followed subsequently. Histological analysis was performed using light microscopy. For the quantification of Nissl staining, the images were processed using ImageJ software. Initially, “Color Deconvolution” was applied via the image menu to isolate the specific staining components. The resulting single-color image was converted to 8-bit grayscale. Segmentation was performed using the “Threshold” tool (Image > Adjust > Threshold), where the intensity range (0–255) was manually adjusted to accurately select the stained regions. Following the generation of a binary image, the “Watershed” option was applied to prevent overlapping cellular structures. After defining the scale, the “Analyze Particles” function was employed to quantify the Integrated Density.

Immunohistochemistry

Cryosections were taken on slides, permeabilized with 0.3% Triton X-100 in Phosphate Buffered Saline (PBS), and then blocked to prevent nonspecific binding. Next, the sections were incubated with primary antibodies specific to the target antigen at 4 °C overnight. The primary antibodies used were: MBP (1:2000, ab7349, Abcam), GFAP (1:1000, PA1–10004, Thermo Fisher Scientific), and NG2 (1:100, PA5–100235, Thermo Fisher Scientific). Incubation with fluorophore-coupled secondary antibodies, Goat anti-Rat Alexa Fluor 555 (1:1000, A-21434, Thermo Fisher Scientific), Goat anti-Chicken Alexa Fluor 488 (1:1000, ab150173, Abcam), and Goat anti-Rabbit Alexa Fluor 647 (1:1000, ab150079, Abcam), was done for 1 h at room temperature. DAPI (4′,6-diamidino-2-phenylindole) was used for 10 min (1 μg/mL) to stain the nucleus. Finally, the sections were mounted with DPX after rinsing twice more with PBS-Tween 20. Slides were examined with a Leica confocal microscope at the necessary laser wavelength. The fluorescence intensity was measured using the ImageJ software program. At first, the RGB measurement was chosen from the ImageJ plugin. Subsequently, the mean value was chosen for each individual marker from the measured window.

Western Blot Analysis

Brain hippocampal tissues (approximately 50–60 mg) were collected for Western blot, and protein extraction was performed by using RIPA buffer. Protein concentration was determined by using the BCA method. Subsequently, equal amount of proteins (30 μg) were loaded onto SDS-PAGE gels and transferred to a PVDF membrane. Then, blocking was done at room temperature for 1 h with 5% BSA to prevent nonspecific binding followed by incubation with primary antibodies against target proteins overnight at 4 °C: p-LIMK1 (1:750, ab194798, Abcam), OLIG 2 (1:1500, PA5–85734, Thermo Fisher Scientific), VEGF (1:1000, MA1–16629, Thermo Fisher Scientific), VCAM-1 (1:2000, PA5–86042, Thermo Fisher Scientific), and GAPDH (1:10000, ab8245, Abcam). After washing with TBS-Tween 20, membranes were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase: Goat anti-Rabbit (1:10000, ab6721, Abcam) and Goat anti-Mouse (1:10000, ab205719, Abcam) for 1 h. Protein bands were visualized using an enhanced chemiluminescence solution (Bio-Rad; 170–5061) in GelDoc (Bio-Rad). The densitometric analysis was executed by ImageJ software. The mean gray value was calculated for the purpose of quantification for each marker and the loading controls associated with them.

Statistical Analysis

GraphPad Prism 8.3.0 was utilized to conduct the statistical analysis. One-way ANOVA was used to compare more than one group. In case of two independent variables across multiple groups, two-way ANOVA was performed. A p-value less than 0.05 was considered to be statistically significant. For behavioral studies, a group of n = 8 was used to ensure statistical significance, while a subset of n = 3 was utilized at each time point for molecular studies to ensure the reproducibility and validity of any observed changes.

Glossary

Abbreviations

ANOVA

Analysis of variance

BBB

blood–brain barrier

CA1

Cornu ammonis 1

CC

corpus callosum

CNS

central nervous system

DG

dentate gyrus

DPX

dibutyl phthalate, polystyrene, xylene

DRG

dorsal root ganglion

EAE

experimental autoimmune encephalomyelitis

GFAP

glial fibrillary acidic protein

IHC

immunohistochemistry

LFB

Luxol Fast Blue

LIMK

Lin-11, Isl-1, and Mec-3 kinase

MAG

myelin-associated glycoprotein

MBP

myelin basic protein

MOG

myelin oligodendrocyte glycoprotein

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MS

multiple sclerosis

MYPT1

myosin phosphatase target subunit 1

NG2

neural/glial antigen 2

OLIG 2

oligodendrocyte transcription factor 2

OPC

oligodendrocyte precursor cell

PBS

phosphate-buffered saline

PSP

postsurgical pain

PVDF

polyvinylidene fluoride

RGB

red, green, and blue

RhoA

Ras homologue family member A

RIPA

radioimmune precipitation

ROCK

Rho-associated coiled-coil containing protein kinase

RPM

revolutions per minute

SCI

spinal cord injury

SDS-PAGE

sodium dodecyl-sulfate polyacrylamide gel electrophoresis

SNI

sparing nerve injury

TBS

tris-buffered Saline

VCAM-1

vascular cell adhesion molecule-1

VEGF

vascular endothelial growth factor

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.6c00015.

• Western blot images for all biological replicates showing the expression levels of p-LIMK, OLIG 2, VEGF, and VCAM across acute (weeks 1, 3, 5, and 7) and chronic (weeks 7, 10, 12, and 14) cuprizone time points; Western blot images of p-LIMK and GFAP across weeks 3, 5, and 7 in control, cuprizone-treated, and BMS-5-treated groups; detailed breakdown of the number of animals utilized across the three study phases (acute, chronic, and therapeutic) (PDF)

A.R.: Writing the original draft, Visualization, Investigation, Data curation, Formal analysis. S.P.: Investigation, Data curation. D.G.: Investigation, Data curation, Editing of the manuscript. H.K.: Conceptualization, Supervision, Reviewing, and Finalizing of the manuscript.

The current work was supported by the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India, and NIPER Ahmedabad. This work was also supported by the Anusandhan National Research Foundation under grant number ANRF/ARG/2025/004116/LS received by Dr. H.K.

The authors declare no competing financial interest.